Thymine formation

During thymine formation the coenzyme is oxidized to dihydrofolate, which must be reduced by dihydrofolate reductase to complete the catalytic cycle. A possible mechanistic sequence for thymidylate synthase, an enzyme of known three-dimensional structure,354/418-4213 is given in Fig. 15-21. In the first step (a) a thiolate anion, from the side chain of Cys 198 of the 316-residue Lactobacillus enzyme, adds to the 5 position of the substrate 2 -deoxyuridine monophosphate... 

During thymine formation the coenzyme is oxidized to dihydrofolate, which must be reduced by dihydrofolate reductase to complete the catalytic cycle. A possible mechanistic sequence for thymidylate synthase, an enzyme of known three-dimensional... 

The methylation of the pyrimidine nucleus by one-carbon compounds was in accord with the earlier suggestion that thymine formation in the rat proceeded by direct conversion of C3rtidine or a closely related structure to thymidine (317). Further agreement came from studies in rabbit bone marrow and in Ehrlich ascites tumor cells in vitro, where it was demonstrated that both c rtidine and deoxycytidine (and to a lesser extent uridine and deoxyuridine) stimulated the incorporation of formate-C into DNA thymine (338). 

They have noted that anaerobically grown E. coli ferment thymidine to thymine, formate (or H2 and CO2), acetate, and ethanol. The process is inhibited by phosphate or sulfate—an inhibition attributed to inhibition... 

The mode of action has been a subject for research for a number of years. While it was originally thought that maleic hydrazide replaced uracil in the RNA sequence, it has been deterrnined that the molecule may be a pyrimidine or purine analogue and therefore base-pair formation is possible with uracil and thymine and there exists the probabiHty of base-pair formation with adenine however, if maleic hydrazide occurs in an in vivo system as the diketo species, then there remains the possibiHty of base-pairing with guanine (50). Whatever the mechanism, it is apparent that the inhibitory effects are the result of a shutdown of the de novo synthesis of protein. 

In contrast, the photochemistry of uracil, thymine and related bases has a large and detailed literature because most of the adverse effects produced by UV irradiation of tissues seem to result from dimer formation involving adjacent thymine residues in DNA. Three types of reaction are recognizable (i) photohydration of uracil but not thymine (see Section 2.13.2.1.2), (ii) the oxidation of both bases during irradiation and (iii) photodimer formation. 

The formation of photodimers of the cyclobutane type from thymine (111 R = Me) occurs most effectively when a frozen aqueous solution of the substrate is irradiated. After the independent recognition of such dimers in two laboratories about 1960, it was some six years before the main constituent was identified beyond doubt (66JCS(C)2239) as the cis-syn entity (113), a U-shaped molecule in which the planes of the six-membered rings... 

The yield of hydroxylated products is always very low, and there are usually a number of by-products. For instance, side chains of aromatic nuclei are easily attacked, as shown by the formation of 5-hydroxymethyluracil from thymine. Breslow and Lukens measured both the amount of 3-hydroxyquinoline formed and the quinoline consumed during hydroxylation with Fenton s reagent and EDTA in the presence of several adducts (Table XII). 

O-isopropylidene derivative (57) must exist in pyridine solution in a conformation which favors anhydro-ring formation rather than elimination. Considerable degradation occurred when the 5-iodo derivative (63) was treated with silver fluoride in pyridine (36). The products, which were isolated in small yield, were identified as thymine and l-[2-(5-methylfuryl)]-thymine (65). This same compound (65) was formed in high yield when the 5 -mesylate 64 was treated with potassium tert-hx Xy -ate in dimethyl sulfoxide (16). The formation of 65 from 63 or 64 clearly involves the rearrangement of an intermediate 2, 4 -diene. In a different approach to the problem of introducing terminal unsaturation into pento-furanoid nucleosides, Robins and co-workers (32,37) have employed mild base catalyzed E2 elimination reactions. Thus, treatment of the 5 -tosylate (59) with potassium tert-butylate in tert-butyl alcohol afforded a high yield of the 4 -ene (60) (37). This reaction may proceed via the 2,5 ... 

Finally a few sentences are deserved for the vast area of DNA photochemistry. Thymine dimerization is the most common photochemical reaction with the quantum yield of formation in isolated DNA of all-thymine oligodeoxynucleotides 2-3% [3], Furthermore, a recent study based on femtosecond time-resolved transient absorption spectroscopy showed that thymine dimers are formed in less than 1 ps when the strand has an appropriate conformation [258], The low quantum yield of the reaction in regular DNA is suggested to be due to the infrequency of these appropriate reactive conformations. 

Durbeej B, Eriksson LA (2002) Reaction mechanism of thymine dimer formation in DNA induced by UV light. Photochem Photobiol A 152 95-101... 

Interestingly, one-electron oxidants partly mimic the effects of OH radicals in their oxidizing reactions with the thymine moiety of nucleosides and DNA. In fact, the main reaction of OH radicals with 1 is addition at C-5 that yields reducing radicals in about 60% yield [34, 38]. The yield of OH radical addition at C-6 is 35% for thymidine (1) whereas the yield of hydrogen abstraction on the methyl group that leads to the formation of 5-methyl-(2 -de-oxyuridylyl) radical (9) is a minor process (5%). Thus, the two major differences in terms of product analysis between the oxidation of dThd by one-electron oxidants and that by the OH radical are the distribution of thymidine 5-hydroxy-6-hydroperoxide diastereomers and the overall percentage of methyl oxidation products. 

Large doses of ultraviolet light can damage DNA. In humans this damage is confined to the skin, since, unlike x-rays, ultraviolet light is easily absorbed. The chemical lesion in this case is the formation of dimers between adjacent thymine residues on the same DNA strand. Unless corrected or removed, these dimers will stop DNA synthesis. 

Psoralens can react by two different routes upon photoactivation (Parsons, 1980 Pathak, 1984). The first route is through the well-known photoreaction mechanism that principally involves intercalation within double-stranded DNA or RNA with the formation of adducts with adjacent thymine bases. The furan-side and pyrone-side rings in psoralen both can form cycloaddition products with the 5,6-double bond of thymine to create a crosslink between two DNA strands (Reaction 57) or to a lesser extent, within double-strand regions of RNA. 

There are basically two types of salvage. The first involves attachment of the base to PRPP with the formation of pyrophosphate. This pathway is available for salvage of purines and uracil but not for cytosine or thymine. The other pathway involves attachment of the base to ribose 1-phosphate, which occurs to some extent for most of the purines and pyrimidines. This second pathway requires the presence of specific... 

Oxygen-free reactions of psoralens, when in close proximity to the target, proceed via the first excited states in which the 3,4-and the 4, 5 7r-bonds of the pyrone and furan moieties, respectively, can undergo C4-cyclization reactions with, e.g., unsaturated bonds of lipids, or the C5=C6 double bonds of thymine in DNA. In reactions with DNA the psoralen is believed to intercalate with DNA in the dark. Subsequent irradiation at 400 nm usually leads to furan-side 4, 5 -monoadduct formation, whereas irradiation at 350 nm increases the formation of crosslinks in which the furan and pyrone rings form C4 cycloadducts to thymines on opposite strands [95], Subsequent irradiation of the 4, 5 -monoadducts at 350 nm leads to formation of crosslinks and conversion into pyrone-side 3,4-monoadducts. Shorter wave-... 

The double-helical rope formation in T-10-T was unexpected, since neither T-10-T nor its precursor thymine is chiral, as verified by CD spectroscopy. The authors speculate that photodimerization of two thymine derivatives leads... 

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